Of all proteins expressed by living organisms, proteases are among the most critical in mediating pathways of cell life and death. In fact, the initial interactions between protease and substrate and subsequent cleavage lie at the base of a vast spectrum of essential biological events including thrombosis, coagulation and apoptosis.
Dysregulated proteolysis, or imbalance between proteases and antiproteases, has been searched intensively based on the suspicion that it could be a key factor in many pathologies where proteases have been involved such as cancer, autoimmune diseases, inflammation and infectious diseases. Different studies done with antiproteolytic agents in cancer and inflammatory disease (such as rheumatoid arthritis and emphysema) models have also shown interesting outcome improvement, strengthening the antiproteolytic therapy and the role of balance between proteases and antiproteases.
For example, in prostate cancer, which is one of the most common diagnosed cancers in American men, proteases are believed to play a pivotal role in the malignant behaviour of cancer cells including rapid tumor growth, invasion, and metastasis.
Human glandular kallikrein (hK2) protein is a trypsin-like serine protease expressed predominantly in the prostate epithelium. Firstly isolated from human seminal plasma, hK2 has recently emerged as a diagnostic marker for prostate cancer (Deperthes et al. 1995 “Isolation of prostatic kallikrein hK2, also known as hGK-1, in human seminal plasma” Biochim Biophys Acta 1245, 311-6).
Beside its role as marker, its proteolytic activities suggest that hK2 could contribute to cancer progression. Several potential functions for this enzyme have been proposed, including the activation of urokinase-type plasminogen activator and inactivation of plasminogen activator inhibitor-1, activation of pro-PSA, degradation of fibronectin and degradation of insulin-like growth factor binding protein (IGF-BP) (for review see Cloutier et al., 2004 “Development of recombinant inhibitors specific to human kallikrein 2 using phase-display selected substrates” Eur J Biochem 3, 607-13).
It has recently been shown that kallikrein hK2 can form a specific complex with a protease inhibitor, known as PI-6, in cancers and particularly in prostate cancer. Based on the discovery of this specific complex, U.S. Pat. Nos. 6,284,873 and 6,472,143 provide a diagnostic method for determining the presence or absence of cancer or tissue necrosis.
Taking into account its prostate tissue specific expression and the involvement of all its potential substrates in cancer development, hK2 is also considered as a potential therapeutic target (Darson et al. 1997 “Human glandular kallikrein 2 (hK2) expression in prostatic intraepithelial neoplasia and adenocarcinoma: a novel prostate cancer marker” Urology 49, 857-62). Therefore, the development of specific and long-lasting protease inhibitors and especially kallikrein inhibitors would be useful.
These Protease inhibitor candidates can be selected among the serpin (serine protease inhibitors) family, which is a large family of proteins implicated in the regulation of complex physiological processes. These proteins of about 45 kDa can be subdivided into two groups, one being inhibitory and the other non-inhibitory.
Serpins contain an exposed flexible reactive-site loop or reactive-serpin loop (RSL), which is implicated in the interaction with the putative target proteinase. Following the binding to the enzyme and cleavage of the P1-P1′ scissile bond of the RSL, a covalent complex is formed (Huntington et al. 2000 “Structure of a serpin-protease complex shows inhibition by deformation” Nature 407, 923-6). Formation of this complex induces a major conformational rearrangement and thereby traps irreversibly the target protease. The inhibitory specificity of serpins is largely attributed to the nature of the residues at P1-P′1 positions and the length of the RSL. Changing the RSL domain or the reactive site of serpins is one approach to understand the inhibitory process between a serpin and an enzyme and to develop specific inhibitors (Dufour et al. 2001 “The contribution of arginine residues within the P6-P1 region of alpha 1-antitrypsin to its reaction with furin” J Biol Chem 276, 38971-9 and Plotnick et al. 2002 “The effects of reactive site location on the inhibitory properties of the serpin alpha(1)-antichymotrypsin” J Biol Chem 277, 29927-35).
Several serpins such as protein C inhibitor, α2 antiplasmin, antithrombin-III, α1-antichymotrypsin (ACT), or protease inhibitor 6 have been identified as hK2 inhibitors (Saedi et al. 2001 “Human kallikrein 2 (hK2), but not prostate-specific antigen (PSA), rapidly complexes with protease inhibitor 6 (PI-6) released from prostate carcinoma cells” Int J Cancer 94, 558-63). The relatively slow complex formation between hK2 and ACT is mainly attributed to residues Leu 358-Ser 359 at P1-P′1 positions of the RSL, an unfavourable peptide bond for this trypsin-like enzyme.
Up to now, only selections of new kallikrein inhibitors, which specifically inhibit plasma kallikrein, and use thereof in therapeutic and diagnostic methods have been disclosed (patents U.S. Pat. No. 6,057,287, U.S. Pat. No. 6,333,402, U.S. Pat. No. 5,994,125, and U.S. Pat. No. 5,795,865). However, these patents describe the production of inhibitors that are homologous to bovine pancreatic trypsin inhibitor Kunitz domains, and especially proteins that are homologous to liproprotein-associated coagulation inhibitor (LACI) Kunitz domains, which specifically inhibit plasma kallikreins.
Besides being specific for plasma kallikrein, these inhibitors are quite small molecules and bind to plasma kallikrein in a reversible manner. One of the major drawback of this approach is that the use of proteins inhibiting their targets in a reversible manner bears the risk that decomplexation of the protease restores its activity.
Therefore, one advantage of using larger inhibitors, as described herein, is that this leads to the formation of covalent complexes which inhibits the protease target in an irreversible manner. A further advantage of the present invention is that large covalent complexes are known to be quickly eliminated from circulation.